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A CRISPR /LbCas12a‐based method for highly efficient multiplex gene editing in Physcomitrella patens
Author(s) -
Pu Xiaojun,
Liu Lina,
Li Ping,
Huo Heqiang,
Dong Xiumei,
Xie Kabin,
Yang Hong,
Liu Li
Publication year - 2019
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.14478
Subject(s) - crispr , biology , trans activating crrna , physcomitrella patens , genome editing , genetics , gene , computational biology , genome , cas9 , gene targeting , crispr interference , mutant
Summary Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR) /Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNA s (cr RNA s) in a single vector. However, the activity and efficiency of CRISPR /Cas12a in the non‐vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature cr RNA can target multiple loci simultaneously in P. patens with high efficiency via co‐delivery of LbCas12a and a cr RNA expression cassette in vivo . The mutation frequencies induced by CRISPR /LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss‐of‐function mutants of multiple genes from different gene families.